The overall goal of the proposed research is to use a combination of molecular and genetic methods to determine the basis for immunity in a model system, Drosophila melanogaster. Recent studies suggest that insects and mammals employ similar strategies to battle bacterial infections. For example, injection of Drosophila larvae with bacteria or lipopolysaccharide induces the appearance of bactericidal proteins. One of these, hemolin, belongs to the immunoglobulin superfamily. It binds to the bacterial cell surface and is thought to mediate a subsequent cellular response. Most of the remaining insect bactericidal proteins fall within 3 groups: cecropins, defensins and attacins. These groups of proteins bind different components of the bacterial cell wall and membrane, and effect synergistic destruction of invading bacteria. Vertebrate defensins have been isolated from neutrophils and macrophages, while cecropins have been found in pig intestine. Recent results suggest that the production of the Drosophila bactericidal peptides is mediated by a regulatory factor cell Dif, which is related to the NF-kappaB/Rel family of proteins. NF-kappaB plays a key role in mammalian immunity and inflammatory response. It is hoped that a detailed characterization of Dif and the signaling pathways will provide basic insights into immunity and acute phase response in human. The research plan includes four specific aims: (i) generate and characterize Dif mutants; (ii) analyze regulatory interactions between Dif and target promoters controlling the expression of immunity genes; (iii) characterize the signaling pathway that regulates Dif activity in response to infection or injury; and, (iv) determine whether acquired immunity is mediated by Dif autoregulation.